Many drug candidates and natural products are flanked with structural features such as carboxylic acids, amines, dianionic phosphates and polyhydroxylated aromatic rings, etc. that may limit their therapeutic potential in vivo. Such features often require temporary protection with a prodrug to improve the absorption, distribution, metabolism and excretion (ADME) properties of a pharmacologically active compound within the body.
One example is Tenofovir (TFV), which is an acyclic nucleoside with anti-viral activity against human immunodeficiency virus (HIV), hepatitis B virus (HBV), and herpes simplex type-2 virus (HSV-2). TFV structurally resembles 2′,3′-dideoxyadenosine which lacks the requisite 3′ hydroxyl moiety necessary for DNA polymerization and triggers obligate chain termination upon incorporation of tenofovir diphosphate (TFVdpp) into the growing viral DNA strand. A common structural feature amongst acyclic nucleosides (e.g. TFV, adefovir, cidofovir, ganciclovir, etc.) is a catabolically stable phosphonate linkage that permanently affixes the phosphonate to the acyclic sugar linker and nucleobase. This serves to prevent undesirable chemical and enzymatic hydrolysis and bypasses the initial phosphorylation to the monophosphate, which is the kinetic bottleneck during the conversion of conventional nucleosides to their active triphosphate (De Clercq et al., Nature reviews. Drug discovery, 2005, 4, 928).
The dianionic character of TFV and other acyclic nucleosides at physiological pH restricts diffusion across the plasma membrane resulting in rapid renal clearance and depreciated bioavailability and antiviral activity. When orally administered to mice, the bioavailability of TFV is approximately 2% and that of adefovir has been reported to be <1% in monkeys and 8-11% in rats (see Kearney et al., J. Clin. Pharmacokinet., 2004, 43, 595, Balzarini et al. AIDS, 1991, 5, 21. Starrett et al., J. Med. Chem., 1994, 37, 1857). These undesirable properties can be ameliorated by masking the anionic phosphonic acid with various prodrugs that alter the pharmacokinetic profile of the parent nucleoside, enhance cellular permeability, and improve bioavailability. Several eclectic prodrug strategies have been developed for this purpose.
The clinically-approved prodrug formulation of TFV is tenofovir disoproxil fumarate (TDF), manufactured by Gilead Sciences under the trade name Viread®, which features two isopropyloxymethyl carbonate masking units esterified to the phosphonate that relies on an esterase-activated cleavage mechanism to liberate TFV following successful delivery to the target tissue. The installation of two isopropyl carbonate esters increases the oral bioavailability of TFV to 25%, dramatically enhances tissue distribution and improves biological stability. However, the ubiquitous distribution of esterases renders a significant fraction of TDF susceptible to premature hydrolysis resulting in systemic exposure to TFV, a known nephrotoxin, potentially causing undesirable side effects (see Karras et al. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 2003, 36, 1070). Continuous administration of TDF has been reported to induce lactic acidosis, Fanconi syndrome, acute renal failure, and bone loss, all of which stem from mitochondrial toxicity (see Fernandez et al., AIDS Res. Treat.; 2011: 354908 and Coca & Perazella, American Journal of the Medical Sciences, 2002, 324(6):342-344).
Other candidates surfaced in clinical trials are Tenofovir-Alafenamide (TAF) and Hexadecyloxypropyl-Tenofovir (CMX-157), as illustrated in FIG. 1. TAF is an isopropylalaninyl phenyl ester that requires two disparate enzymes for prodrug release: carboxyesterase and cathepsin A. The latter enzyme, cathepsin A, is a serine protease localized almost exclusively to lysosomal endosomes and ensures selective intracellular delivery of TFV. TAF is currently approved in the clinic and demonstrates little to no nephrotoxicity and more potent antiviral activity than TDF at 1/10th the dose. Painter et al. reported evaluations of CMX-157 as a potential treatment for HIV type 1 and HBV infections (Antimicrob. Agents Chemother., 2007, 51, 3505-3509). CMX-157 relies on the catalytic activity of an intracellular hydrolase-phospholipase C and/or sphingomylenase to liberate TFV within the cytosol. Available preliminary data indicates CMX-157 is well-tolerated and achieves significant concentrations of TFVdpp up to one week after a single 400 mg dose, indicating the potential for a convenient, once-a-week dosing regimen. However, CMX-157 has made little progression through the clinical trial pipeline since the completion of Phase I in 2011.
Gosselin and collaborators previously examined dithioethanol (DTE) conjugates to mediate the delivery of adefovir, AZT, and 3′-deoxyuridine (ddU) to HIV-1 infected cell lines in vitro. U.S. Pat. No. 6,020,482 reports phosphotriester type biologically active compounds and also see U.S. Pat. Nos. 6,555,676, 7,902,202, and 8,871,785.
Conjugation of bis(DTE) to adefovir increases the HIV-1 activity of the parent nucleoside by ten-fold and confers exceptional stability (t1/2>24 h) at pH 2, pH 7.4, culture medium, and human gastric juice. However, these conjugates rapidly degrade in human serum (t1/2<5 min) which significantly limits their clinical utility. A proposed cleavage mechanism for these masking groups is illustrated in FIG. 2. Reduction of compound (i) releases β-mercaptoethanol and metastable intermediate (ii) that spontaneously collapses on the thioethanol linker to generate thiirane and the free nucleoside (iii) (when R=H). It was speculated that the β-mercaptoethyl linker may be a precursor for thiirane which has been implicated in the decomposition of S-acyl-2-thioethyl (SATE) and dithioethanol (DTE) prodrugs. Of note, SATE prodrugs also pass through common intermediate (ii) following hydrolysis of an S-acyl moiety via non-specific carboxyesterases to liberate the target nucleoside. The apparent instability of the disulfide linkage in serum has stalled efforts to advance this technology forward and the mutagenic potential of thiirane has precluded the clinical use of SATE and DTE-bound nucleosides. References cited herein are not an admission of prior art.
Thus, there is a need to identify improvements and exploit alternative prodrug strategies to enhance intracellular delivery of pharmacologically active compounds, such as useful nucleosides like tenofovir.